Ecto-5′-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5′-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5′-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5′-AMP. EPEC infection triggered a release of ecto-5′-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5′-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5′-nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate (α,β-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5′-AMP. In addition, α,β-methylene-ADP and zinc blocked the ability of 5′-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5′-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5′-nucleotidase, such as α,β-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.     

Effect of the L- or D-aspartate on ecto-5′nucleotidase activity and on cellular viability in cultured neurons: participation of the adenosine A<Subscript>2A</Subscript> receptors

Summary. Glutamate increases the extracellular adenosine levels, an important endogenous neuromodulator. The neurotoxicity induced by glutamate increases the ecto-5′-nucleotidase activity in neurons, which produces adenosine from AMP. L- and D-aspartate (Asp) mimic most of the actions of glutamate in the N-methyl-D-aspartate (NMDA) receptors. In the present study, both amino acids stimulated the ecto-5′-nucleotidase activity in cerebellar granule cells. MK-801 and AP-5 prevented the L- and D-Asp-evoked activation of ecto-5′-nucleotidase. Both NMDA receptor antagonists prevented completely the damage induced by L-Asp, but partially the D-Asp-induced damage. The antagonist of adenosine A_2A receptors (ZM 241385) prevented totally the L- Asp-induced cellular death, but partially the neurotoxicity induced by D-Asp and the antagonist of adenosine A₁ receptors (CPT) had no effect. The results indicated a different involvement of NMDA receptors on the L- or D-Asp-evoked activation of ecto-5′-nucleotidase and on cellular damage. The adenosine formed from ecto-5′-nucleotidase stimulation preferentially acted on adenosine A_2A receptor which is probably co-operating with the neurotoxicity induced by amino acids.     

Adenosine,but not guanosine,protects vaginal epithelial cells from Trichomonas vaginalis cytotoxicity

Trichomonas vaginalis causes the most common non-viral sexually transmitted disease worldwide. The cytoadherence and cytotoxicity upon the vaginal epithelial cells are crucial for the infection. Extracellular nucleotides are released during cell damage and, along with their nucleosides, can activate purinoceptors. The opposing effects of nucleotides versus nucleosides are regulated by ectonucleotidases. Herein we evaluated the hemolysis and cytolysis induced by T. vaginalis, as well as the extracellular nucleotide hydrolysis along with the effects mediated by nucleotides and nucleosides on cytotoxicity. In addition, the gene expression of purinoceptors in host cells was determined. The hemolysis and cytolysis exerted by all T. vaginalis isolates presented positive Pearson correlation. All T. vaginalis isolates were able to hydrolyze nucleotides, showing higher NTPDase than ecto-5′-nucleotidase activity. The most cytotoxic isolate, TV-LACM6, hydrolyzes ATP, GTP with more efficiency than AMP and GMP. The vaginal epithelial cell line (HMVII) expressed the genes for all subtypes of P1, P2X and P2Y receptors. Finally, when nucleotides and nucleosides were tested, the cytotoxic effect elicited by TV-LACM6 was increased with nucleotides. In contrast, the cytotoxicity was reversed by adenosine in presence of EHNA, but not by guanosine, contributing to the understanding of the purinergic signaling role on T. vaginalis cytotoxicity.     

Adenosine reduces reactive oxygen species and interleukin-8 production by <Emphasis Type="Italic">Trichomonas vaginalis-</Emphasis>stimulated neutrophils

Trichomonas vaginalis is a flagellated protozoan that affects the human urogenital tract causing 276.4 million new infections a year. The parasite elicits a vaginal mucosal infiltration of immune cells, especially neutrophils which are considered to be primarily responsible for cytological change observed at the infection site as well as the major contributor in the inflammatory response against the parasite. Extracellular nucleotides and their nucleosides are signaling compounds involved in several biological processes, including inflammation and immune responses. Once in the extracellular space, the nucleotides and nucleosides can directly activate the purinergic receptors. Herein, we investigated the involvement of purinergic signaling on the production of reactive oxygen species (ROS) and cytokines by T. vaginalis-stimulated neutrophils. Parasites were able to induce an increase in ROS and IL-8 levels while they did not promote IL-6 secretion or neutrophil elastase activity. Adenine and guanine nucleotides or nucleosides were not able to modulate ROS and cytokine production; however, when T. vaginalis-stimulated neutrophils were incubated with adenosine and adenosine deaminase inhibitor, the levels of ROS and IL-8 were significantly reduced. These immunosuppressive effects were probably a response to the higher bioavailability of adenosine found in the supernatant as result of inhibition of enzyme activity. The involvement of P1 receptors was investigated by immunofluorescence and A1 receptor was the most abundant. Our data show that the influence of purinergic signaling, specifically those effects associated with adenosine accumulation, on the modulation of production of proinflammatory mediators by T. vaginalis-stimulated neutrophils contribute to the understanding of immunological aspects of trichomoniasis.     

Activity and expression of ecto-5′-nucleotidase/CD73 are increased by thyroid hormones in vascular smooth muscle cells

Extracellular nucleotides ATP, ADP, AMP and adenosine are well known signaling molecules of the cardiovascular system that are involved in several physiological processes: cell proliferation, platelet aggregation, inflammatory processes and vascular tonus. The levels of these molecules are controlled by ecto-NTPDases and ecto-5′-nucleotidase/CD73 (ecto-5′-NT/CD73) actions, which are responsible for the complete ATP degradation to adenosine. The thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), play important roles in the vascular system promoting vasodilatation. Here we investigated the influence of thyroid hormones on the enzyme cascade that catalyzes the interconversion of purine nucleotides in vascular smooth muscle cells (VSMC). Exposure of VSMCs to 50nM T₃ or T₄ did not change ATP and ADP hydrolysis significantly. However, the same treatment caused an increase of 75% in AMP hydrolysis, which was time-dependent but dose-independent. Moreover, T₃ treatment significantly increased ecto-5′-NT/CD73 mRNA expression, which suggests a genomic effect of this hormone upon ecto-5′-NT/CD73. In addition to the importance of the ecto-5′-NT in cell proliferation and differentiation, its overexpression could result in higher extracellular levels of adenosine, an important local vasodilatator molecule.     

Regulation of the Ecto-Nucleotidase Pathway in Rat Hippocampal Nerve Terminals

Ecto-nucleotidases play a pivotal role in terminating the signalling via ATP and in producing adenosine, a neuromodulator in the nervous system. We have now investigated the pattern of adenosine formation with different concentrations of extracellular ATP in rat hippocampal nerve terminals. It was found that adenosine formation is delayed with increasing concentrations of ATP. Also, the rate of adenosine formation increased sharply when the extracellular concentrations of ATP + ADP decrease below 5 M, indicating that ATP/ADP feed-forwardly inhibit ecto-5-nucleotidase allowing a burst-like formation of adenosine possibly designed to activate facilitatory A_2A receptors. Initial rate measurements of ecto-5-nucleotidase in hippocampal nerve terminals, using IMP as substrate, showed that ATP and ADP are competitive inhibitors (apparent K_i of 14 and 4 M). In contrast, in hippocampal immunopurified cholinergic nerve terminals, a burst-like formation of adenosine is not apparent, suggesting that channelling processes may overcome the feed-forward inhibition of ecto-5-nucleotidase, thus favouring A₁ receptor activation.     

Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling

Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y₁ and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase, P2Y₁, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic calcium responses and cellular volume.     

High expression and activity of ecto-5′-nucleotidase/CD73 in the male murine reproductive tract

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615–628, 2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main enzyme responsible for the generation of adenosine from AMP, namely the ecto-5′-nucleotidase (CD73). The enzyme was identified by immunological techniques and by in situ enzymatic assays, including inhibition experiments with α,β-methylene-ADP, a specific CD73 inhibitor. High levels of ecto-5′-nucleotidase were detected in testes in association with both germinal and somatic cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5′-nucleotidase that, in concurrence with NTPDases, may impact male fertility.     

Physiological roles for ecto-5’-nucleotidase (CD73)

Nucleotides and nucleosides influence nearly every aspect of physiology and pathophysiology. Extracellular nucleotides are metabolized through regulated phosphohydrolysis by a series of ecto-nucleotidases. The formation of extracellular adenosine from adenosine 5′-monophosphate is accomplished primarily through ecto-5′-nucleotidase (CD73), a glycosyl phosphatidylinositol-linked membrane protein found on the surface of a variety of cell types. Recent in vivo studies implicating CD73 in a number of tissue protective mechanisms have provided new insight into its regulation and function and have generated considerable interest. Here, we review contributions of CD73 to cell and tissue stress responses, with a particular emphasis on physiologic responses to regulated CD73 expression and function, as well as new findings utilizing Cd73-deficient animals.     

Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis

Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.     

Prostatic acid phosphatase, a neglected ectonucleotidase

Two recent papers reveal that the soluble and secreted prostatic acid phosphatase, an enzyme that has long served as a diagnostic marker for prostate cancer, has a membrane-bound splice variant. This enzyme exhibits ecto-5′-nucleotidase activity, is widely distributed, and implicated in the formation of chronic pain. While prostatic acid phosphatase hydrolyzes phosphomonoesters other than 5′-nucleoside monophosphates these novel data suggest that, in addition to ecto-5′-nucleotidase and the alkaline phosphatases, prostatic acid phosphatase must be taken into account in future studies on extracellular adenosine production.     

Superoxide Anion Production by Human Neutrophils Activated by Trichomonas vaginalis

Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O₂^.-) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.     

A calcium channel blocker activates both ecto-5(')-nucleotidase and NO synthase in HUVEC

Since amlodipine, a long-acting Ca channel blocker, increases both NO and adenosine production in canine hearts, we investigated that amlodipine activates both ecto-5(')-nucleotidase responsible for adenosine production and NO synthase (NOS) for NO production in human umbilical venous endothelial cells (HUVECs), and its cellular signaling. We measured activities of ecto-5(')-nucleotidase and NOS in HUVECs in the condition with additions of xanthine (100 microM)+xanthine oxidase (1.6 x 10(-3)U/ml) in the presence or absence of amlodipine (1 x 10(-9)-1 x 10(-6)M). Amlodipine increased both ecto-5(')-nucleotidase and NOS activities. Xanthine+xanthine oxidase deactivated both NOS and ecto-5(')-nucleotidase, and amlodipine increased both activities of NOS and ecto-5(')-nucleotidase by 117+/-33% and 48+/-6%, respectively. Amlodipine phosphorylated p38MAP kinase and that an inhibitor of p38MAP kinase inhibited the amlodipine-induced activation of both NOS and ecto-5(')-nucleotidase. Furthermore, amlodipine increased both adenosine and NO production in the canine ischemic hearts. We concluded that amlodipine activates both NOS and ecto-5(')-nucleotidase via p38MAP kinase in vitro and enhances both NO and adenosine production in vivo.     

Atorvastatin accelerates extracellular nucleotide degradation in human endothelial cells

HMG-CoA reductase inhibitors (statins) exert pleiotropic effects in the cardiovascular system beyond its cholesterol-lowering action. We aimed to investigate how atorvastatin affects extracellular nucleotide degradation in human endothelial cells, as increased activity of this pathway would facilitate conversion of pro-inflammatory nucleotides into anti-inflammatory adenosine. Primary cultures of human endothelial cells were treated with 1 μM, 10 μM and 100 μM atorvastatin for 24 h. Enzyme assays were performed as well as intact cell studies, to evaluate capacity of cells to degrade ATP to adenosine. Atorvastatin significantly increased ATP breakdown and adenosine formation in the medium of intact cells in a dose-dependent manner. The activities of ATPase, ADPase and ecto-5′-nucleotidase (eN) in cell homogenates following Atorvastatin treatment were also increased while no change was observed in the lactate dehydrogenase activity. We suggest a new mechanism of protective effect of atorvastatin by activation of endothelial enzymes involved in extracellular nucleotide degradation in human endothelial cells.     

Ectonucleoside triphosphate diphosphohydrolases and ecto-5′-nucleotidase in purinergic signaling: how the field developed and where we are now

Geoffrey Burnstock will be remembered as the scientist who set up an entirely new field of intercellular communication, signaling via nucleotides. The signaling cascades involved in purinergic signaling include intracellular storage of nucleotides, nucleotide release, extracellular hydrolysis, and the effect of the released compounds or their hydrolysis products on target tissues via specific receptor systems. In this context ectonucleotidases play several roles. They inactivate released and physiologically active nucleotides, produce physiologically active hydrolysis products, and facilitate nucleoside recycling. This review briefly highlights the development of our knowledge of two types of enzymes involved in extracellular nucleotide hydrolysis and thus purinergic signaling, the ectonucleoside triphosphate diphosphohydrolases, and ecto-5′-nucleotidase.

     

Ectonucleotidases in the kidney

Members of all four families of ectonucleotidases, namely ectonucleoside triphosphate diphosphohydrolases (NTPDases), ectonucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-5′-nucleotidase and alkaline phosphatases, have been identified in the renal vasculature and/or tubular structures. In rats and mice, NTPDase1, which hydrolyses ATP through to AMP, is prominent throughout most of the renal vasculature and is also present in the thin ascending limb of Henle and medullary collecting duct. NTPDase2 and NTPDase3, which both prefer ATP over ADP as a substrate, are found in most nephron segments beyond the proximal tubule. NPPs catalyse not only the hydrolysis of ATP and ADP, but also of diadenosine polyphosphates. NPP1 has been identified in proximal and distal tubules of the mouse, while NPP3 is expressed in the rat glomerulus and pars recta, but not in more distal segments. Ecto-5′-nucleotidase, which catalyses the conversion of AMP to adenosine, is found in apical membranes of rat proximal convoluted tubule and intercalated cells of the distal nephron, as well as in the peritubular space. Finally, an alkaline phosphatase, which can theoretically catalyse the entire hydrolysis chain from nucleoside triphosphate to nucleoside, has been identified in apical membranes of rat proximal tubules; however, this enzyme exhibits relatively high K _m values for adenine nucleotides. Although information on renal ectonucleotidases is still incomplete, the enzymes’ varied distribution in the vasculature and along the nephron suggests that they can profoundly influence purinoceptor activity through the hydrolysis, and generation, of agonists of the various purinoceptor subtypes. This review provides an update on renal ectonucleotidases and speculates on the functional significance of these enzymes in terms of glomerular and tubular physiology and pathophysiology.     

Enzymes involved in metabolism of extracellular nucleotides and nucleosides: Functional implications and measurement of activities

Abstract

Extracellular nucleotides and nucleosides mediate diverse signaling effects in virtually all organs and tissues. Most models of purinergic signaling depend on functional interactions between distinct processes, including (i) the release of endogenous ATP and other nucleotides, (ii) triggering of signaling events via a series of nucleotide-selective ligand-gated P2X and metabotropic P2Y receptors as well as adenosine receptors and (iii) ectoenzymatic interconversion of purinergic agonists. The duration and magnitude of purinergic signaling is governed by a network of ectoenzymes, including the enzymes of the nucleoside triphosphate diphosphohydrolase (NTPDase) family, the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, ecto-5′-nucleotidase/CD73, tissue-nonspecific alkaline phosphatase (TNAP), prostatic acid phosphatase (PAP) and other alkaline and acid phosphatases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). Along with “classical” inactivating ectoenzymes, recent data provide evidence for the co-existence of a counteracting ATP-regenerating pathway comprising the enzymes of the adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK/NME/NM23) families and ATP synthase. This review describes recent advances in this field, with special emphasis on purine-converting ectoenzymes as a complex and integrated network regulating purinergic signaling in such (patho)physiological states as immunomodulation, inflammation, tumorigenesis, arterial calcification and other diseases. The second part of this review provides a comprehensive overview and basic principles of major approaches employed for studying purinergic activities, including spectrophotometric P_i-liberating assays, high-performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) analyses of purine substrates and metabolites, capillary electrophoresis, bioluminescent, fluorometric and electrochemical enzyme-coupled assays, histochemical staining, and further emphasizes their advantages, drawbacks and suitability for assaying a particular catalytic reaction.     

Stimulation of an α1-adrenergic receptor downregulates ecto-5′ nucleotidase activity on the apical membrane of RPE cells

The purines ATP and adenosine play an important role in the communication between the photoreceptors and the retinal pigment epithelium (RPE). While the RPE is known to release ATP into subretinal space, the source of extracellular adenosine is unclear. In other tissues, ecto-nucleotidases mediate the consecutive dephosphorylation of ATP to AMP, and AMP is converted to adenosine by ecto-5′ nucleotidase (CD73). This study identifies ecto-5′ nucleotidase on RPE cells and investigates modulation of enzyme activity. The RPE was the most active site of 5′AMP dephosphorylation in the posterior rat eye. The ecto-5′ nucleotidase inhibitor αβmADP prevented the production adenosine by the apical membrane of the bovine RPE. Cultured human ARPE-19 cells expressed mRNA and protein for ecto-5′ nucleotidase. The production of phosphate from 5′AMP by ARPE-19 cells was inhibited by αβmADP, but the ecto-alkaline phosphatase inhibitor levamisole had no effect. Degradation of 5′AMP was blocked by norepinephrine, epinephrine and phenylephrine, with inhibition by antagonists prazosin and corynanthine implicating the α1 adrenergic receptor. The block of enzyme activity by norepinephrine was rapid, occurring within 1 min, and was similar at both 4 and 37°C, consistent with cleavage of the enzyme from its GPI anchor. HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath. In the apical face of the bovine-RPE eyecup, norepinephrine reduced the production of phosphate from 5′AMP, suggesting that both receptor and enzyme face sub-retinal space. In conclusion, RPE cells express ecto-5′ nucleotidase, with activity on the apical membrane, and stimulation of α-1 adrenergic receptors downregulates activity. As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time.